Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

Joachim M Gerhold,Oskar Engberg,Ian J Holt,Johannes N Spelbrink,Aurelio Reyes,Madis Lõhmus,Helen M Cooper,Helga Van Rennes,Şirin Cansiz-Arda,Alberto Sanz

doi:10.1038/srep15292

Abstract

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

Highlights

Since nucleoids involve protein-DNA and protein-RNA interactions to facilitate mitochondrial DNA (mtDNA) maintenance and gene expression they likely are very dynamic[5]
We have repeated the flotation experiments, analyzed the lipid profile of the different fractions from such flotation gradients and show that the majority of the mitochondrial cholesterol is concentrated in the same fractions as the Twinkle-containing nucleoids (Fig. 1a and SFig. 1a)
We propose that the Twinkle-containing mammalian nucleoids attach to a membrane structure that is highly enriched in cholesterol, is held together by ATAD3 and is found in association with ER-mitochondrial junctions

Summary

Introduction

Since nucleoids involve protein-DNA and protein-RNA interactions to facilitate mtDNA maintenance and gene expression they likely are very dynamic[5]. According to this view nucleoids exist in several populations, which differ in their protein components depending on functional requirements such as replication, translation and repair. The altered expression of ATAD3 perturbed mtDNA topology, as well as mitochondrial protein synthesis[10,11] These observations prompted us to propose that ATAD3 supports nucleoids and mitochondrial ribosomes on the IM11. ATAD3 has been shown to regulate intestinal fat storage in nematodes[13] and cholesterol channeling in a steroidogenic cell line[14] Taken together, these observations suggest that ATAD3 is involved in the biogenesis or structural maintenance of mitochondrial membranes. ER-associated mitochondrial division has been spatially linked to nucleoids in yeast[26], and in mammalian cells nucleoids have been observed to often lie adjacent to dynamin related protein 1 (Drp1)[27,28], which controls mitochondrial fission

Methods

Results

Conclusion